CN102753964A - Mass spectrometric determination of non-derivatized, non-metabolized vitamin d - Google Patents

Abstract

The invention relates to the detection of non-metabolized vitamin D. In a particular aspect, the invention relates to methods for detecting underivatized non-metabolized vitamin D by mass spectrometry.

Description

The mass spectroscopy of the vitamin D of non-that derive, non-metabolism

Invention field

The present invention relates to the quantitative measurment of the vitamin D of non-metabolism.Aspect concrete, the present invention relates to method through the vitamin D of the non-metabolism of tandem mass spectrum method quantitative measurement.

Background of invention

Vitamin D is at calcium (Ca ²⁺) have the essential nutrients of important physiological effect in just the regulating of stable state.Vitamin D can in skin, from the beginning form through being exposed to daylight or it can be from dietary ingestion.There are two kinds of form vitamin Ds: vitamin D ₂(ergocalciferol) and vitamin D ₃(Vitamin D3).Vitamin D ₃Be by the from the beginning synthetic form of animal.It still joins at the dairy products of produced in usa and the common fill-in in some food.Diet and inherent synthetic vitamin D ₃Must experience metabolic activation to produce bioactive metabolin.In the mankind, vitamin D ₃The initial step that activates mainly occurs in the liver and comprises that hydroxylation is to form intermedium metabolin 25-hydroxvcholecalciferol (calcifediol; 25OHD ₃).Calcifediol is a vitamin D in the circulation ₃Principal mode.Round-robin 25OHD then ₃Transform by kidney, form 1, the 25-dihydroxyvitamin D ₃(calcitriol; 1,25 (OH) ₂D ₃), it is considered to have the vitamin D of high bioactivity usually ₃Metabolin.

Vitamin D ₂From fungi and plant origin.Many OTC dietary supplements (dietary supplements) contain ergocalciferol (vitamin D ₂) rather than Vitamin D3 (vitamin D ₃).Vitamin D2 (Drisdol)---in efficient prescription form of the obtainable unique vitamin D of the U.S.---is prepared with ergocalciferol.Vitamin D ₂Experience and vitamin D in the mankind ₃Similar metabolic activation approach forms metabolin 25OHD ₂With 1,25 (OH) ₂D ₂Vitamin D ₂And vitamin D ₃Be assumed in for a long time among the mankind and biologically equating; Yet proposing the vitamin D of these two kinds of forms, nearest report on biologically active and bioavailability, can there are differences (Armas et.al., (2004) J.Clin.Endocrinol.Metab.89:5387-5391).

The measurement of vitamin D---non-activity vitamin D precursor---seldom takes place in clinical setting.On the contrary, 25-hydroxy-vitamin D ₃, 25-hydroxy-vitamin D ₂With the serum levels of total 25-hydroxy-vitamin D (" 25OHD ") be the useful indicant of the effectiveness of vitamin D nutrition condition and some novel vitamin D analogues.The measurement of 25OHD is generally used for the diagnosis and the processing of disorder of calcium metabolism.In this respect, the alkaline phosphatase of low-level 25OHD indication and disease such as hypocalcemia, hypophosphatemia, secondary hyperparathyroidism, rising, be grown up in the relevant vitamin D deficiency of rickets among malacosteon and the children.In the patient of doubtful poisoning by vitamin D, other illness that the 25OHD of elevated levels is different from this illness to cause hypercalcinemia.

1,25 (OH) ₂The measurement of D also is used for clinical setting.Some morbid state can be through 1,25 (OH) of cyclical level ₂The D reflection, for example kidney disease and renal failure usually cause low-level 1,25 (OH) ₂D.1,25 (OH) of elevated levels ₂D can indicate excessive parathormone maybe can indicate the lymthoma of some disease such as sarcoidosis or some type.

The detection of vitamin D metabolism thing is through using 25OHD ₂And 25OHD ₃The radiommunoassay of common special antibody is accomplished.Because present does not differentiate 25OHD respectively based on immunologic mensuration ₂And 25OHD ₃So without recourse to other test, the source of any nutritional deficiency of vitamin D can not be determined.Delivered the report that openly utilizes mass spectroscopy to detect the method for specified vitamin D metabolin.In some reports, the vitamin D metabolism thing was derived before mass spectroscopy, but in other report, they are not like this.The U.S. Patent Application Serial 11/946765 that Holmquist for example, et al.2007 submitted at Dec 28; Yeung B, et al., J Chromatogr.1993,645 (1): 115-23; Higashi T, et al., Steroids.2000,65 (5): 281-94; Higashi T, et al., Biol Pharm Bull.2001,24 (7): 738-43; Higashi T, et al., J Pharm Biomed Anal.2002,29 (5): 947-55; Higashi T, et al., Anal.Biochanal Chem, 2008,391:229-38; And Aronov, et al., Anal Bioanal Chem, 2008, the open method that detects various vitamin D metabolism things through the metabolin of before mass spectroscopy, deriving of 391:1917-30.The method that detects the vitamin D metabolism thing do not derive is at Clarke, et al., the U.S. Patent Application Serial 11/101 that on April 6th, 2005 submitted to; 11/386 of submission on March 21st, 166 and 2006; In 215, and Singh, et al.; Be in the news in the U.S. Patent Application Serial 10/977,121 that on October 24th, 2004 submitted to.Also disclose report, it openly uses Cookson type reagent, particularly 4-phenyl-1; 2,4-triazoline-3, [2-(6 for 5-diketone (PTAD) and 4-; 7-dimethoxy-4 '-methyl-3-oxo-3,4-dihydro-quinoxaline base (dihydroquinoxalyl)) ethyl]-1,2; 4-triazoline-3, the vitamin D that 5-diketone (DMEQ-TAD) carries out ₃Derive.Referring to Aberhart, J, et al., J.Org.Chem.1976,41 (12): 2098-2102 and Kamao, M, et al., J Chromatogr.B2007,859:192-200.

Summary of the invention

The present invention provides the method through the amount of the vitamin D of one or more (non-metabolism) forms in the mass spectroscopy test sample that comprises tandem mass spectrometry.On the one hand, derived through quilt before the analytical reagent composition from the vitamin D of sample.Second aspect is not being derived through before the analytical reagent composition from the vitamin D of sample.

In some embodiments of first aspect, this method may further comprise the steps: the vitamin D that the Cookson type in the sample is derived is being fit to experience ionization source under one or more conditions through the detectable precursor ion of mass spectroscopy of generation; (ii) broken at least a said precursor ion is to produce one or more through the detectable fragmention of mass spectroscopy; (iii) through one or more the amount in mass spectrometric determination precursor ion and the fragmention; The amount of the ion of measuring in (iii) in step is associated with the amount of vitamin D in the sample.In these methods, sample is being enough to produce before experience Cookson type derivative reagent under the condition of the vitamin D that the Cookson type derives in step (i).The vitamin D of in some embodiments, the Cookson type being derived experienced extraction column and analytical column before ionization.In related embodiment, analytical column can be high performance liquid chromatography (HPLC) post.

In some embodiments of first aspect, this method may further comprise the steps: (i) make sample experience turbulent flow liquid chromatography (TFLC); (ii) make the vitamin D of deriving be fit to experience ionization source under one or more conditions of generation through the detectable ion of mass spectroscopy from the Cookson type of sample; The amount of the vitamin D ion of (iii) deriving through one or more Cookson types of mass spectrometric determination; The amount of the vitamin D ion that the Cookson type measured in (iii) in step derives is associated with the amount of vitamin D in the sample.In these methods, sample is enough to produce before experience Cookson type derivative reagent under the condition of the vitamin D that the Cookson type derives in step (i) in sample.In some embodiments, afterwards but step makes sample experience high performance liquid chromatography (HPLC) before (ii) in step (i).

In some embodiments, Cookson type derivative reagent is a 4-phenyl-1,2,4-triazoline-3,5-diketone (PTAD).In some embodiments in these embodiments, one or more precursor ions comprise that one or more are selected from mass-to-charge ratio (m/z) is 572.4 ± 0.5 and 560.4 ± 0.5 ion.In some embodiments in these embodiments, one or more fragmentions comprise that mass-to-charge ratio (m/z) is 298.1 ± 0.5 ion.

Comprise vitamin D at vitamin D ₂Embodiment in, the vitamin D ion that one or more Cookson types are derived can comprise that mass-to-charge ratio (m/z) is that 572.3 ± 0.5 precursor ion and mass-to-charge ratio (m/z) are 298.1 ± 0.5 fragmention.Comprise vitamin D at vitamin D ₃Embodiment in, the vitamin D ion that one or more Cookson types are derived can comprise that mass-to-charge ratio (m/z) is that 560.3 ± 0.5 precursor ion and mass-to-charge ratio (m/z) are 298.1 ± 0.5 fragmention.Comprise vitamin D at vitamin D ₂And D ₃Embodiment in, one or more precursor ions can comprise that mass-to-charge ratio (m/z) is 572.4 ± 0.5 vitamin D ₂Precursor ion and m/z are 560.4 ± 0.5 vitamin D ₃Precursor ion; One or more fragmentions can comprise that m/z is 298.1 ± 0.5 vitamin D ₂Fragmention and m/z are 298.1 ± 0.5 vitamin D ₃Fragmention.

In some embodiments of second aspect, the present invention provides and need not ionization and detect the vitamin D of deriving, through the method for the amount of vitamin D in the tandem mass spectrometry working sample.In some embodiments, vitamin D comprises vitamin D ₂These methods may further comprise the steps: make the vitamin D from sample ₂Be fit to produce one or more through mass spectroscopy detectable being selected under the condition of precursor ion that mass-to-charge ratio (m/z) is 397.2 ± 0.5 or 379.2 ± 0.5 ion experience ionization source; Broken at least a said precursor ion is to produce one or more through the detectable fragmention of mass spectroscopy; (iii) through mass spectrometric determination step (i) with (ii) in the amount of one or more ions of generation; (iv) make the (iii) middle vitamin D of measuring of step ₂Vitamin D in the existence of ion and the sample ₂Existence be associated.In these methods, if broken precursor ion comprises that m/z is 397.2 ± 0.5 ion, fragmention comprises that one or more are selected from the ion that m/z is 159.0 ± 0.5,146.9 ± 0.5,133.1 ± 0.5 and 121.0 ± 0.5 ion so.In these methods, if broken precursor ion comprises that m/z is 379.2 ± 0.5 ion, fragmention comprises that one or more are selected from the ion that m/z is 283.2 ± 0.5,187.3 ± 0.5,175.2 ± 0.5 and 159.0 ± 0.5 ion so.In some embodiments, can be before ionization with vitamin D from sample ₂The experience extraction column is like SPE (SPE) post or turbulent flow liquid chromatography (THLC) post.In some embodiments, will be before ionization from the vitamin D of sample ₂Undergoing analysis post in addition is like high performance liquid chromatography (HPLC) post.

In other embodiment of second aspect, vitamin D comprises vitamin D ₃These methods may further comprise the steps: (i) make the vitamin D from sample ₃Be fit to produce one or more through mass spectroscopy detectable being selected under the condition of precursor ion that mass-to-charge ratio (m/z) is 385.2 ± 0.5 or 367.2 ± 0.5 ion experience ionization source; (ii) broken at least a said precursor ion is to produce one or more through the detectable fragmention of mass spectroscopy; (iii) through mass spectrometric determination step (i) with (ii) in the amount of one or more ions of generation; (iv) make the (iii) middle vitamin D of measuring of step ₃Vitamin D in the existence of ion and the sample ₃Existence be associated.In these methods, if broken precursor ion comprises that m/z is 385.2 ± 0.5 ion, fragmention comprises that one or more are selected from the ion that m/z is 159.0 ± 0.5,147.0 ± 0.5,133.1 ± 0.5 and 107.1 ± 0.5 ion so.If broken precursor ion comprises that m/z is 367.2 ± 0.5 ion, fragmention comprises that one or more are selected from m/z is 172.2 ± 0.5,145.0 ± 0.5 and 119.1 ± 0.5 ion so.In some embodiments, can be before ionization with vitamin D from sample ₃The experience extraction column is like SPE (SPE) post or turbulent flow liquid chromatography (THLC) post.In some embodiments, will be before ionization from the vitamin D of sample ₃Undergoing analysis post in addition is like high performance liquid chromatography (HPLC) post.

In method described herein, mass spectroscopy can be a tandem mass spectrometry.In utilizing the embodiment of tandem mass spectrometry, tandem mass spectrometry can---for example comprise that multiple reaction monitoring, precursor ion scanning or product ion scan---through any method known in the art to carry out.

In utilizing the embodiment of extraction column, extraction column can be SPE (SPE) post; Like turbulent flow liquid chromatography (TFLC) post.In some embodiments, it utilizes two or more in extraction column, analytical column and the ionization source, and the component of utilization can connect to allow the sample preparation and the analysis of robotization with on-line mode.

The Cookson type derivative reagent that is used for some embodiment can be selected from 4-phenyl-1,2,4-triazoline-3,5-diketone (PTAD), 4-methyl isophthalic acid; 2,4-triazoline-3,5-diketone (MTAD), 4-[2-(6,7-dimethoxy-4 '-methyl-3-oxo-3; 4-dihydro-quinoxaline base) ethyl]-1,2,4-triazoline-3,5-diketone (DMEQTAD), 4-(4-nitrobenzophenone)-1; 2,4-triazoline-3,5-diketone (NPTAD) and 4-ferrocenyl methyl isophthalic acid; 2,4-triazoline-3,5-diketone (FMTAD) with and isotope-labeled variant.In a preferred embodiment, Cookson type derivative reagent is a 4-phenyl-1,2,4-triazoline-3,5-diketone (PTAD) or its isotope-labeled variant.In some preferred implementation, Cookson type derivative reagent is a 4-phenyl-1,2,4-triazoline-3,5-diketone (PTAD) or its isotope-labeled variant.

As used herein, term " vitamin D " refers to the analog of vitamin D of vitamin D or the synthetic non-metabolism of any or multiple naturally occurring non-metabolism.This is relative with the vitamin D metabolism thing of identifying through the particular chemical modification that takes place in the metabolic process (for example, 25-hydroxy-vitamin D and 1 α, 25-dihydroxyvitamin D).The vitamin D of non-metabolism also can be known as " trophism " vitamin D to be different from the form of metabolism.Mention the form that the vitamin D of not specifying metabolisable form is meant non-metabolism.

As used herein, " deriving " instigates two molecular reactions to form new molecule.Therefore, derivating agent is and the agent of another substance reaction with derived material.For example, 4-phenyl-1,2,4-triazoline-3,5-diketone (PTAD) are the derivative reagents that can react the vitamin D of deriving with formation PTAD-with vitamin D.

As used herein, the title of derivative form vitamin D comprises the expression about the character of deriving.For example, vitamin D ₂The PTAD derivant be represented as the PTAD-vitamin D ₂(or the PTAD-vitamin D of deriving ₂).

As used herein, " Cookson type derivating agent " is that 4-is substituted 1,2,4-triazoline-3,5-dione compounds.Exemplary Cookson type derivating agent comprises 4-phenyl-1,2,4-triazoline-3,5-diketone (PTAD), 4-methyl isophthalic acid, 2; 4-triazoline-3,5-diketone (MTAD), 4-[2-(6,7-dimethoxy-4 '-methyl-3-oxo-3,4-dihydro-quinoxaline base) ethyl]-1,2; 4-triazoline-3,5-diketone (DMEQTAD), 4-(4-nitrobenzophenone)-1,2,4-triazoline-3; 5-diketone (NPTAD) and 4-ferrocenyl methyl isophthalic acid, 2,4-triazoline-3,5-diketone (FMTAD).In addition, the isotope-labeled variant of Cookson type derivating agent capable of using in some embodiments.For example, ¹³C ₆-PTAD isotopic variations weighs 6 mass units and can be used for some embodiments than normal PTAD.The deriving of vitamin D metabolism thing that carries out through Cookson type reagent can carry out through any appropriate method.Referring to, for example, Holmquist, et al., the U.S. Patent Application Serial 11/946765 that on Dec 28th, 2007 submitted to; Yeung B, et al., J Chromatogr.1993,645 (1): 115-23; Higashi T, et al., Steroids.2000,65 (5): 281-94; Higashi T, et al., Biol Pharm Bull.2001,24 (7): 738-43; Higashi T, et al., J Pharm Biomed Anal.2002,29 (5): 947-55; Higashi T, et al., Anal.Biochanal Chem, 2008,391:229-38; And Aronov, et al., Anal Bioanal Chem, 2008,391:19 17-30.

Vitamin D can refer to the vitamin D of one or more forms, like vitamin D ₂And/or vitamin D ₃Comprise in the embodiment of multivitamin D form that at sample multivitamin D form can be by while ionization.For example, in some embodiments, in identical sample, measure vitamin D ₂And vitamin D ₃Amount.In these embodiments, (derive or do not derive) vitamin D ₂And vitamin D ₃Can be by while ionization.

As being used for detecting simultaneously two or more amounts from the analyte of sample, term " simultaneously " refers to the data of the amount of two or more analytes in the reflection sample that from identical sample injection, obtains.The data of each analyte can be successively or parallel acquisition, and this depends on the technical device of utilization.For example, can the single sample that comprise two kinds of analytes be injected into the HPLC post, HPLC post every kind of analyte of wash-out one by one causes successively analyte being introduced mass spectrometer then.For the purpose of this paper is measured in these two kinds of analytes every kind amount simultaneously, because two kinds of analytes are all produced by the identical sample that injection gets into HPLC.

Vitamin D can find in the circulation of animal and/or can be produced like animal by biosome.Like this, sample can, for example, from the patient; That is, the living person of sex obtains, and in clinical setting, presents the diagnosis, prognosis or the treatment that self are used for disease or situation.Preferred sample can be a biological sample; Particularly biologicfluid sample such as serum or blood plasma.When sample during from the mankind, the method that this paper appears can be used for measuring the amount that is present in the vitamin D in the sample.

In some preferred implementation of this paper disclosed method, mass spectroscopy is carried out with the kation mode.Alternatively, mass spectroscopy is carried out with the negative ion mode.Various ionization sources comprise for example APCI (APCI), laser diode thermal desorption (LDTD) or electrospray ionization (ESI), can be used for embodiment of the present invention.In some embodiments, utilize APCI or LDTD to measure the vitamin D metabolism thing with the kation mode.

In a preferred embodiment, one or more detectable separately interior marks are provided in the sample, also in sample, have measured target amount in this.In these embodiments; With the interested analyte of all or part that exists in the sample (one or more) and interior mark (one or more) ionization with produce multiple in mass spectrometer detectable ion, and detect one or more ions from every kind of generation through mass spectroscopy.Preferably, interior mark (one or more) is a vitamin D ₂-[6,19,19]- ²H ₃, vitamin D ₂-[26,26,26,27,27,27]- ²H ₆, vitamin D ₃-[6,19,19]- ²H ₃And vitamin D ₃-[26,26,26,27,27,27]- ²H ₆In one or more.

Before with the processing of Cookson type derivative reagent (if applicable) or any purifying, can in sample, provide one or more to mark in detectable separately from the analyte (one or more) of sample.In these embodiments, mark can be derived and/or purifying together with the endogenous vitamin D in one or more, and in this case, interior target ion that derive and/or purifying detects through mass spectroscopy.In these embodiments, the existence or the amount of the ion that produces from interested analyte can be relevant with the existence or the amount of analytes of interest analytes the sample.In some embodiments, interior mark can be the isotope-labeled form of vitamin D, like vitamin D ₂-[6,19,19]- ²H ₃, vitamin D ₂-[26,26,26,27,27,27]- ²H ₆, vitamin D ₃-[6,19,19]- ²H ₃And vitamin D ₃-[26,26,26,27,27,27]- ²H ₆

In the exemplary interior mark of listing above can being directed against each produces detectable ion in mass spectrometer.Exemplary spectrum to mark in the several exemplary produces is described in embodiment 8 and 9, and is presented among Fig. 6-7,9-10,12-13 and the 15-16.

As used herein, when analyzing with mass-spectrometric technique, with respect to unlabelled molecule, " isotope labeling " produces mass shift in the molecule of mark.The example of the mark that is fit to comprise deuterium ( ²H), ¹³C with ¹⁵N.For example, vitamin D ₂-[6,19,19]- ²H ₃And vitamin D ₃-[6,19,19]- ²H ₃Has the vitamin D of ratio respectively ₂And vitamin D ₃The quality of high about 3 mass units.Isotope labeling can be mixed the one or more positions in molecule, and one or more isotope labelings can be used on the identical isotope-labeled molecule.

In other embodiments, the amount of vitamin D ion (one or more) can be through measuring with one or more External Reference standard comparisons.Exemplary External Reference standard comprises and is mixed with vitamin D ₂-[6,19,19]- ²H ₃, vitamin D ₂-[26,26,26,27,27,27]- ²H ₆, vitamin D ₃-[6,19,19]- ²H ₃And vitamin D ₃-[26,26,26,27,27,27]- ²H ₆In one or more blank plasma or serum.In the vitamin D of deriving embodiment to be detected, external standard will experience and the identical processing and the analysis of any other sample that will be analyzed usually, be included in before the mass spectrography processing with one or more Cookson type reagent.

In some embodiments, vitamin D ₂And vitamin D ₃Quantitative lower limit (LLOQ) less than 10ng/mL; Preferably less than 5ng/mL; Preferably less than 2ng/mL.

As used herein, unless otherwise indicated, singulative " (a) ", " one (an) ", " said (the) " comprise plural reference.Therefore, for example, mention that " albumen (a protein) " comprises a plurality of protein moleculars.

As used herein, term " purifying (purification) " or " purifying (purifying) " are not meant all materials of from sample, removing except interested analyte (one or more).On the contrary, but purifying refers to other component with respect to the interested detection of analytes of interfere in the sample, the process of the amount of one or more interested analytes of enrichment.Sample purifying through various means can allow one or more interfering materials---for example, maybe or possibly not disturb the parent ion of the selection through mass spectroscopy or one or more materials that daughter ion detects---relative minimizing.When this term uses, reduce not requiring relatively with being removed fully through purifying by any material that exists with interested analyte in the material of purifying.

As used herein, term " solid phase extraction " or " SPE " refer to since dissolving or be suspended in the solution (that is moving phase) component to solution through or the affinity of the solid (that is solid phase) of flowing through and chemical mixture is divided into the process of each component.In some instances, when moving phase through or when flowing through solid phase, the not desired components of moving phase can be retained in the solid phase, causes the purifying of analyte in the moving phase.In other instance, analyte can be retained in the solid phase, the not desired components that allows moving phase through or the solid phase of flowing through.In these instances, then second moving phase is used for the analyte that keeps eluted from solid phase and is used for further handling or analyzing.SPE---comprises TFLC---can be through single-mode or the operation of mixed mode mechanism.Mixed mode mechanism is utilized ion-exchange and hydrophobic reservation in same post; For example, the solid phase of mixed mode SPE post can show reinforcing yin essence ion-exchange and hydrophobic reservation; Maybe can show strong cation exchange and hydrophobic reservation.

As used herein, term " chromatography " refer to since when flowing through fixing liquid phase or solid phase the difference of chemical entities distribute and the chemical mixture of liquid or gas-entrained be divided into the process of each component.

As used herein, term " liquid chromatography " or " LC " refer to the process of one or more components selection property retardances of fluid solution when fluid filters through the post of the meticulous material that separates or through capillary channel equably.Retardance is by when fluid moves with respect to stationary phase (or a plurality of), and each component of the potpourri between one or more stationary phase and the quantity of liquid (that is moving phase) distributes and produces.The instance of " liquid chromatography " comprises reversed phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC) and turbulent flow liquid chromatography (TFLC) (being known as high turbulent flow liquid chromatography (HTLC) or high flux liquid chromatography sometimes).

As used herein; Term " high performance liquid chromatography " or " HPLC " (being known as " high pressure liquid chromatography " sometimes) refer to such liquid chromatography; Wherein through under pressure, forcing flow communication to cross stationary phase, the post of normally closely filling, and increase the degree of separating.

As used herein, term " turbulent flow liquid chromatography " or " TFLC " (being known as high turbulent flow liquid chromatography or high flux liquid chromatography sometimes) refer to a kind of chromatogram of form, and its turbulent flow of utilizing the just determined material through column filling is as the basis of separating.Before passing through analytical reagent composition, TFLC has been applied to prepare the sample that comprises two kinds of unnamed medicines.Referring to, for example, Zimmer et al., J Chromatogr A 854:23-35 (1999); Also referring to, U.S. Patent number 5,968,367,5,919,368,5,795,469 and 5,772,874, it further explains TFLC.Those of ordinary skills understand " turbulent flow ".When fluid flowed at leisure and glibly, this flowed and is known as " laminar flow ".For example, be stratiform with the fluid of low flow velocity through the HPLC post.The movement of particles of fluid is Methodistic in laminar flow, has usually with straight-line particle.In fast speeds, the inertia of water overcomes fluid friction power and turbulization." do not surpass " fluid that is slowed down or departed from by rough surface by rubbing with irregular border fluid in contact.When fluid during with turbulent flows, it flows with whirlpool and whirling motion (or vortex), flows to have more " resistance (drag) " when being stratiform.Can obtain many references be used for auxiliary confirm fluid when flow be stratiform or turbulent flow (for example, Turbulent Flow Analysis: Measurement and Prediction, P.S.Bernard & J.M.Wallace, John Wiley & Sons, Inc., (2000); An Introduction to Turbulent Flow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

As used herein; Term " gas chromatography " or " GC " refer to such chromatogram; Wherein sample mixture evaporation and the carrier gas (like nitrogen or helium) that is injected into through comprising stationary phase the post motion of---being made up of liquid or granular solids---are flowed, and according to compound the affinity of stationary phase is divided into it and respectively forms compound.

As used herein, term " bulky grain post " or " extraction column " refer to comprise the chromatographic column greater than the mean grain size of about 50 μ m.

As used herein, term " analytical column " refers to have enough chromatosheets to cause the chromatographic column of separating substances in the sample, and this material is enough to allow the existence or the amount of determination and analysis thing from the post wash-out.In a preferred embodiment, analytical column contains the particle of the about 5 μ m of diameter.Such post usually is different from " extraction column ", and extraction column has the sample to obtain purifying that from non-material retained, separates or extract material retained and is used for the further purpose of analysis.

As used herein, term " online (on-line) " and " online (inline) " for example as used in " online automated manner " or " online extraction ", refer to not need operator intervention and the program of carrying out.By contrast, as used herein, term " off line (off-line) " need to refer to the program of operator's manual intervention.Therefore, if sample is precipitated, then with the supernatant manual load in automatic sampler, deposition and to load step be off line with subsequently step.In the various embodiments of method, one or more steps can be carried out with online automated manner.

As used herein, term " mass spectroscopy " or " MS " refer to the analytical technology through the Quality Identification compound of compound.MS refers to based on the mass-to-charge ratio of ion or " m/z ", filters, detects and measure the method for ion.The MS technology generally includes (1) ionization compound to form charged compound; (2) molecular weight of the charged compound of detection and calculating mass-to-charge ratio.Compound can detect by ionization with through any suitable method." mass spectrometer " generally includes ionization source and ion detector.Generally speaking, with one or more molecule (s) of interest ionization, and subsequently ion is introduced mass spectrometer, because the associating of magnetic field and electric field, ion depends on that in the space path of quality (" m ") and electric charge (" z ") walks in mass spectrometer.Referring to, for example, name is called the U.S. Patent number 6,204,500 of " Mass Spectrometry From Surfaces "; The name be called " Methods and Apparatus for Tandem Mass Spectrometry " 6,107,623; The name be called " DNA Diagnostics Based On Mass Spectrometry " 6,268,144; The name be called " Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes " 6,124,137; Wright et al., Prostate Cancer and Prostatic Diseases 1999,2:264-76; With Merchant and Weinberger, Electrophoresis 2000,21:1164-67.

As used herein, term " turns round with the negative ion mode " and refers to produce and detect anionic those mass spectroscopy methods.As used herein, term " turns round with the kation mode " and refers to produce and detect cationic those mass spectroscopy methods.

As used herein, term " ionization (ionization) " or " ionization (ionizing) " refer to produce the process of the analyte ions with the net charge that equals one or more electron units.Negative ion is those ions with net negative charge of one or more electron units, and kation is those ions with clean positive charge of one or more electron units.

As used herein, term " electron ionization " or " EI " refer to such method, and wherein the interested analyte of gas phase or vapour phase and electron stream interact.The bump of electronics and analyte produces analyte ions, then can be with this analyte ions experience mass spectroscopy technology.

As used herein, term " chemi-ionization " or " CI " refer to such method, wherein reagent gas (for example ammonium) are carried out electron collision, through the interaction formation analyte ions of reagent gas ion and analyte molecule.

As used herein, term " fast atom bombardment " or " FAB " refer to such method, and wherein energetic atom bundle (Xe or Ar usually) clashes into nonvolatile sample, the molecule that comprises in desorb and the ionized sample.Test specimen is dissolved in liquid matrix such as glycerine, thioglycerin, a position nitrobenzyl alcohol, 18-hat-6 crown ethers, 2-nitrobenzene octyl ether, sulfolane, diethanolamine and the triethanolamine of thickness.The selection of the suitable matrix of compound or sample is an empirical process.

As used herein; Term " matrix assisted laser desorption ionization " or " MALDI " refer to such method; Wherein nonvolatile sample is exposed to laser emission, it is through various ionization approach---comprising photoionization, protonation, deprotonation and bunch decay (cluster the decay)---analyte in desorb and the ionized sample.For MALDI, sample is mixed with endergonic matrix, this matrix promotes the desorb of analyte molecule.

As used herein; Term " surface-enhanced laser desorption ionization " or " SELDI " refer to another kind of method; Wherein nonvolatile sample is exposed to laser emission, it is through various ionization approach---comprising photoionization, protonation, deprotonation and bunch decay (cluster the decay)---analyte in desorb and the ionized sample.For SELDI, usually sample is attached to the surface of preferential one or more interested analytes of reservation.As in MALDI, this process endergonic material also capable of using is to promote ionization.

As used herein, term " electrospray ionization " or " ESI " refer to such method, wherein solution are transmitted along the segment kapillary, and the electromotive force of high plus or minus is applied to capillary end.Make and arrive jet or the spraying that the terminal solution vaporization (atomizing) of pipe becomes the very droplet of solution in the solvent vapo(u)r.This spray pervaporation chamber, it is condensed and makes solvent evaporation preventing by mild heat.Diminish along with dripping, the ammeter surface charge density increased up to such time---and the natural repulsion between the same electric charge causes that ion and neutral molecule discharge.

As used herein, term " APCI " or " APCI " refer to the mass spectroscopy method similar with ESI; Yet, interacting through the heteroion in atmospheric pressure occurs in plasma, APCI produces ion.Plasma keeps through spray capillary with to electric discharge between electrodes.Usually the skimmer stage (skimmer stages) of utilizing one group of differential to pump is then extracted mass analyzer usually with ion.The N of drying and preheating ₂The adverse current of gas can be used for improving the removal of solvent.Gas phase ionization among the APCI can more effectively be used to analyze the lower kind of polarity than ESI.

As used herein, term " atmospheric pressure photoionization " or " APPI " refer to a kind of ionization of form, and wherein the ionization mechanism of molecule M is that photonic absorption and electrospray are to form molion M+.Because the photon energy that absorbs is usually just above ionic potential, molion relatively is difficult for disassociation.In many cases, therefore possible analytic sample and need not chromatogram saves plenty of time and cost.Under the situation that has water vapour or proton solvent, molion can be drawn H to form MH+.If M has high proton affinity, this is tending towards taking place.This does not influence quantitative precision, because M+ and MH+ sum are constant.Usually the medical compounds in the proton solvent is viewed as MH+, and non-polar compound such as naphthalene or testosterone form M+ usually.Referring to, for example, Robb et al., Anal.Chem.2000,72 (15): 3653-3659.

As used herein, term " inductively coupled plasma " or " ICP " refer to such method, and wherein sample and partially ionized gas interact so that make most elements atomization and ionization in fully high temperature.

As used herein, term " desorb " refers to get into gas phase from surface removal analyte and/or analyte.Laser diode thermal desorption (LDTD) is such technology, and the sample that wherein comprises analyte is drawn onto gas phase through the laser pulse pyrolysis.Laser light strikes is with the back side of 96 orifice plates of the special preparation of metal base.INFRARED PULSE LASER IRRADIATION base, heat make sample changeover become gas phase.Can gas phase sample inspiration ionization source with the ionization in preparation of gas phase sample, be used for analyzing at mass spectrometer there then.When utilizing LDTD, the ionization of gas phase sample can be accomplished through any suitable technique known in the art, as through the ionization (for example passing through APCI) with corona discharge.

As used herein, term " field desorption " refers to such method, wherein nonvolatile test specimen is put on the ionization surface, and highfield is used for to produce analyte ions.

As used herein, term " selected ion monitoring " is the detection mode of mass spectrometer, wherein has only the ion of narrow relatively mass range, and---common about mass unit---is detected.

As used herein, " many reactive modes " is known as " reaction monitoring of selection " sometimes, is the detection mode of mass spectrometer, and wherein precursor ion is optionally detected with one or more fragmentions.

As used herein, term " quantitative lower limit (lower limit of quantification) ", " quantitative lower limit (lower limit of quantitation) " or " LLOQ " refer to measure the quantitatively significant point that becomes.In the response of the analyte of this LOQ is discernible, discrete with reproducible, has relative standard deviation (RSD%) and 80% to 120% accuracy less than 20%.

As used herein, term " detection limit value " or " LOD " are such points, and be big in the more relative uncertainty of this point measurement value.LOD is such point, and at this point, value surpasses the uncertainty relevant with its measurement and is defined in three times of RSD of zero-dose mean value.

As used herein, " amount " of analyte is often referred to the absolute value of detectable analyte quality in the reflection sample volume in the humoral sample.Yet amount is also considered the relative quantity compared with another amount of analyte.For example, the amount of analyte can be the amount greater than contrast that is present in the analyte in the sample usually or normal level in the sample.

As used herein, mention the quantitative measurment that does not comprise that mass of ion is measured, term " about " 10% the indicated value that refers to add deduct.The mass spectroscopy instrument can slightly change when measuring the quality of given analyte.Term " about " is at context+/-0.50 atomic mass unit of the mass-to-charge ratio of the quality of ion or ion.

Above-described summary of the invention is unrestricted, and based on following detailed description of the present invention with based on claim, further feature of the present invention and advantage will be tangible.

The accompanying drawing summary

Figure 1A-C shows the PTAD-vitamin D respectively ₂, the PTAD-vitamin D ₃With the PTAD-vitamin D ₃-[6,19,19]- ²H ₃The exemplary chromatogram of (interior mark).Details are discussed in embodiment 3.

Fig. 2 A and 2B show the vitamin D in the blood serum sample of measuring through the method for describing among the embodiment 3 ₂And vitamin D ₃Exemplary calibration curve.

Fig. 3 shows vitamin D ₂And vitamin D ₃The coefficient of variation to the figure of concentration.Details are described in embodiment 5.

Fig. 4 shows vitamin D in the different samples matrix ₃The result of the comparative studies of analyzing.Details are described in embodiment 11.

Fig. 5 A shows vitamin D ₂The exemplary Q1 scanning spectrum of ionization (covering about 300 to 450 m/z scope).Fig. 5 B shows the vitamin D with m/z of about 397.2 ₂The exemplary products ionic spectrum (covering about 100 to 400 m/z scope) that precursor ion is cracked.Fig. 5 C shows the vitamin D with m/z of about 379.2 ₂The exemplary products ionic spectrum (covering about 100 to 400 m/z scope) that precursor ion is cracked.Details are described in embodiment 14.

Fig. 6 A shows vitamin D ₂-[6,19,19]- ²H ₃The exemplary Q1 scanning spectrum of ion (covering about 300 to 450 m/z scope).Fig. 6 B shows the vitamin D with m/z of about 400.2 ₂– [6,19,19]- ²H ₃The exemplary products ionic spectrum (covering about 100 to 400 m/z scope) that precursor ion is cracked.Fig. 6 C shows the vitamin D with m/z of about 382.2 ₂-[6,19,19]- ²H ₃The exemplary products ionic spectrum (covering about 100 to 400 m/z scope) that precursor ion is cracked.Details are described in embodiment 14.

Fig. 7 A shows vitamin D ₂-[26,26,26,27,27,27]- ²H ₆The exemplary Q1 scanning spectrum of ion (covering about 300 to 450 m/z scope).Fig. 7 B shows the vitamin D with m/z of about 403.2 ₂-[26,26,26,27,27,27]- ²H ₆The exemplary products ionic spectrum (covering about 100 to 400 m/z scope) that precursor ion is cracked.Fig. 7 C shows the vitamin D with m/z of about 385.2 ₂-[26,26,26,27,27,27]- ²H ₆The exemplary products ionic spectrum (covering about 100 to 400 m/z scope) that precursor ion is cracked.Details are described in embodiment 14.

Fig. 8 A shows vitamin D ₃The exemplary Q1 scanning spectrum of ionization (covering about 300 to 450 m/z scope).Fig. 8 B shows the vitamin D with m/z of about 385.2 ₃The exemplary products ionic spectrum (covering about 100 to 400 m/z scope) that precursor ion is cracked.Fig. 8 C shows the vitamin D with m/z of about 367.2 ₃The exemplary products ionic spectrum (covering about 100 to 400 m/z scope) that precursor ion is cracked.Details are described in embodiment 14.

Fig. 9 A shows vitamin D ₃– [6,19,19]- ²H ₃The exemplary Q1 scanning spectrum of ion (covering about 300 to 450 m/z scope).Fig. 9 B shows the vitamin D with m/z of about 388.2 ₃– [6,19,19]- ²H ₃The exemplary products ionic spectrum (covering about 100 to 400 m/z scope) that precursor ion is cracked.Fig. 9 C shows the vitamin D with m/z of about 370.2 ₃– [6,19,19]- ²H ₃The exemplary products ionic spectrum (covering about 100 to 400 m/z scope) that precursor ion is cracked.Details are described in embodiment 14.

Figure 10 A shows vitamin D ₃– [26,26,26,27,27,27]- ²H ₆The exemplary Q1 scanning spectrum of ion (covering about 300 to 450 m/z scope).Figure 10 B shows the vitamin D with m/z of about 391.2 ₃– [26,26,26,27,27,27]- ²H ₆The exemplary products ionic spectrum (covering about 100 to 400 m/z scope) that precursor ion is cracked.Figure 10 C shows the vitamin D with m/z of about 373.2 ₃– [26,26,26,27,27,27]- ²H ₆The exemplary products ionic spectrum (covering about 100 to 400 m/z scope) that precursor ion is cracked.Details are described in embodiment 14.

Figure 11 A shows the PTAD-vitamin D ₂The exemplary Q1 scanning spectrum of ion (covering about 500 to 620 m/z scope).Figure 11 B shows the PTAD-vitamin D with m/z of about 572.2 ₂The exemplary products ionic spectrum (covering about 250 to 350 m/z scope) that precursor ion is cracked.Details are described in embodiment 15.

Figure 12 A shows the PTAD-vitamin D ₂– [6,19,19]- ²H ₃The exemplary Q1 scanning spectrum of ion (covering about 500 to 620 m/z scope).Figure 12 B shows the PTAD-vitamin D with m/z of about 575.2 ₂– [6,19,19]- ²H ₃The exemplary products ionic spectrum (covering about 250 to 350 m/z scope) that precursor ion is cracked.Details are described in embodiment 15.

Figure 13 A shows the PTAD-vitamin D ₂– [26,26,26,27,27,27]- ²H ₆The exemplary Q1 scanning spectrum of ion (covering about 500 to 620 m/z scope).Figure 13 B shows the PTAD-vitamin D with m/z of about 578.2 ₂-[26,26,26,27,27,27]- ²H ₆The exemplary products ionic spectrum (covering about 250 to 350 m/z scope) that precursor ion is cracked.Details are described in embodiment 15.

Figure 14 A shows the PTAD-vitamin D ₃The exemplary Q1 scanning spectrum of ion (covering about 500 to 620 m/z scope).Figure 14 B shows the PTAD-vitamin D with m/z of about 560.2 ₃The exemplary products ionic spectrum (covering about 250 to 350 m/z scope) that precursor ion is cracked.Details are described in embodiment 15.

Figure 15 A shows the PTAD-vitamin D ₃-[6,19,19]- ²H ₃The exemplary Q1 scanning spectrum of ion (covering about 500 to 620 m/z scope).Figure 15 B shows the PTAD-vitamin D with m/z of about 563.2 ₃-[6,19,19]- ²H ₃The exemplary products ionic spectrum (covering about 250 to 350 m/z scope) that precursor ion is cracked.Details are described in embodiment 15.

Figure 16 A shows the PTAD-vitamin D ₃– [26,26,26,27,27,27]- ²H ₆The exemplary Q1 scanning spectrum of ion (covering about 500 to 620 m/z scope).Figure 16 B shows the PTAD-vitamin D with m/z of about 566.2 ₃– [26,26,26,27,27,27]- ²H ₆The exemplary products ionic spectrum (covering about 250 to 350 m/z scope) that precursor ion is cracked.Details are described in embodiment 15.

Detailed Description Of The Invention

The method of vitamin D in the measuring samples is described.The mass spectrometry method of vitamin D in detection and the quantitative sample more specifically, is described.This method Cookson type capable of using reagent like PTAD, produces the vitamin D of deriving.Yet, in certain methods, do not use derivating agent, detect the vitamin D of not deriving through mass spectroscopy ₂And/or vitamin D ₃

This method extraction chromatography technology capable of using, like turbulent flow liquid chromatography (TFLC), connexus spectrometry (MS) method vitamin D that do not derive or that derive ₂And/or vitamin D ₃Purifying provides vitamin D in detection and the quantitative sample thus ₂And/or vitamin D ₃High throughput assay systems.Alternatively, in certain methods, it is optional to sample analysis that chromatogram---comprises extraction chromatography---.In these methods, make the vitamin D of not deriving or derive with LDTD ₂/ or vitamin D ₃Ionization.Preferred implementation is particularly suitable for the application in the quantitative big clinical labororatory of robotization vitamin D.

The suitable test specimen that is used for the inventive method comprises any test specimen that can contain interested analyte.In some preferred implementations, sample is a biological sample; That is, from any biogenetic derivation, like the sample of acquisitions such as animal, cell culture, organ cultures.In some preferred implementation, sample is from mammal, like acquisitions such as dog, cat, horses.Preferred especially mammal is a primate, and most preferably sex is human.Preferred sample comprises body fluid such as blood, blood plasma, serum, saliva, cerebrospinal fluid or tissue sample; Preferred blood plasma (comprising EDTA and heparin blood plasma) and serum; Serum most preferably.Such sample can, for example, from the patient; That is, the living person, sex obtains, and in clinical setting, presents the diagnosis, prognosis or the treatment that self are used for disease or situation.

The present invention also considers the vitamin D quantitative determination reagent kit.The kit of vitamin D quantitative measurement can comprise the kit that comprises the composition that this paper provides.For example, kit can comprise the Cookson type reagent and the isotope-labeled interior mark of wrappage and the amount of measuring---with the amount of enough at least one mensuration.Usually, kit will comprise that also instructions (for example, being included on paper or the electronic media) with tangible form record is used for using the reagent of the packing that the vitamin D quantitative measurement uses.

The calibration that is used for embodiment of the present invention preferably utilizes the matrix similar with the sample matrices of expecting to prepare with the QC storehouse.

Be used for the specimen preparation of mass spectrophotometry

Be used for the preparation of mass spectrophotometry; Vitamin D can be through any method known in the art; For example comprise; Liquid chromatography, filtration, centrifugal, thin-layer chromatography (TLC), comprise the electrophoresis of Capillary Electrophoresis, the affine separation in comprising that immunity is affine and being separated in, comprise the extraction method of ethyl acetate or methanol extraction and utilize chaotropic agent or any combination of above or similar approach is carried out enrichment with respect to one or more other components (for example protein) in the sample.

Protein precipitation is a kind of preparation test specimen, particularly biologic test sample, like the method for serum or blood plasma.Method of purifying protein is well known in the art, for example, Polson et al., Journal of Chromatography B2003,785:263-275 has described the protein precipitation technology that is suitable for the inventive method.Protein precipitation can be used for from sample, removing most of protein, stays vitamin D in supernatant.Can sample is centrifugal with separating liquid supernatant from the protein of deposition; Alternatively can be with sample filtering to remove the protein of deposition.Can supernatant that obtain or filtrating be directly applied to analytical reagent composition then; Or be applied to liquid chromatography and analytical reagent composition subsequently alternatively.In some embodiments, sample, like blood plasma or serum, can be through hybrid protein matter deposition/LLE and by purifying.In these embodiments, sample is mixed with methyl alcohol, ethyl acetate and water, and with the potpourri vortex that produces and centrifugal.With the supernatant that produces remove, bone dry and reconstruct in acetonitrile (reconstituted).Then can be with the vitamin D of purifying with any Cookson type reagent, preferred PTAD or its isotope-labeled variant are derived.

Another method of the sample purifying that can before mass spectroscopy, use is liquid chromatography (LC).Some method that comprises the liquid chromatography of HPLC relies on slow relatively laminar-flow technique.Traditional HPLC analyzes and relies on column filling, and wherein the laminar flow of the sample through post is the basis of from sample, separating interested analyte.Those skilled in the art will understand, and the separation in such post is the LC that comprises HPLC, instrument and the post that diffusion process and the vitamin D that can select to be fit to derive use.Chromatographic column generally includes medium (that is packing material) to promote the separation (that is classification) of chemical part.Medium can comprise small particle, maybe can comprise the integral material with duct.The surface of medium generally includes bonding surface, and it interacts to promote separating of chemical part with various chemical parts.Suitable bonding surface is hydrophobic bonding surface as alkyl linked, cyanic acid bonding surface or high-purity silica surface.Alkyl linked surface can comprise the alkyl of C-4, C-8, C-12 or C-18 bonding.In a preferred embodiment, post is high-purity silica column (like Thermo Hypersil Gold Aq post).Chromatographic column comprises the outlet that is used to receive the inlet of sample and is used to discharge the effluent of the sample that comprises classification.Can sample directly be provided to inlet, or from extraction column, like online SPE cylinder or TFLC extraction column.

In one embodiment, can sample be applied to the LC post at inlet, with solvent or solvent mixture wash-out, and in the outlet discharge.Can select different solvent modes to be used for the interested analyte of wash-out (or a plurality of).For example, liquid chromatography gradient mode capable of using, etc. degree mode or changeable (promptly mix) mode carry out.During chromatogram, the separation of material is accomplished through the selection of variable such as eluent (also being known as " moving phase "), type of elution, gradient condition, temperature etc.

In some embodiments, through sample is kept by column material at interested analyte reversiblely, can be and be applied to post under the condition that one or more other materials are not retained with the analyte purifying.In these embodiments, the first moving phase condition capable of using when interested analyte is kept by post, in case non-material retained was washed, the second moving phase condition capable of using subsequently is to remove material retained from post.Alternatively, but through comparing one or more other materials at interested analyte under the moving phase condition of differentiated speed wash-out sample is applied to post purity analysis thing.Such operation can be with respect to the amount of one or more other one or more interested analytes of component enrichment of sample.

In a preferred implementation, carry out HPLC with alkyl linked analytical column chromatographic system.In some preferred implementation, use high-purity silicon dioxide post (like Thermo Hypersil Gold Aq post).In some preferred implementation, utilize hplc grade water to carry out HPLC and/or TFLC as Mobile phase B as mobile phase A and HPLC level ethanol.

Through carefully selector valve and connecting tube, can two or more chromatographic columns be connected when needing and need not any manual step so that material passes through to the next one from one.In a preferred embodiment, the selection of valve and pipeline by preprogrammed computer control to carry out essential step.Most preferably, chromatographic system also is connected to detector system with such on-line mode, for example, and the MS system.Therefore, the operator can be put into the pallet of sample in the automatic sampler, and remaining operating under the computer control carried out, and causes the sample of all selections to obtain purifying and analysis.

In some embodiments, extraction column can be used for the purifying of vitamin D metabolism thing before the mass spectroscopy.In such embodiment, utilize the extraction column of catching analyte before the ionization, sample can be extracted, then on second extraction column or analyzing wash-out and chromatography on the HPLC post.For example, can use the packed column of coarsegrain (50 μ m) to realize with the sample extraction of TFLC extraction column.The sample transfer that can the wash-out of this post be gone out then is used for being further purified before the mass spectroscopy to the HPLC analytical column.Because the step that comprises in these chromatographic programs can connect with the robotization mode, so can the needs that operator in the analyte purge process participates in be reduced to minimum.These characteristics can cause saving time and cost, and eliminate the possibility of operator error.

In some embodiments, protein precipitation is through hybrid protein matter deposition/LLE, and---comprising methyl alcohol protein precipitation and ethyl acetate/water extraction from serum---realizes.The vitamin D metabolism compound that before the experience extraction column, produces can be derived.Preferably, hybrid protein matter deposition/LLE is connected with on-line mode with extraction column.In a preferred embodiment, extraction column is the C-8 extraction column, like the online extraction column of Cohesive Technologies C8XL (50 μ m granularities, 0.5 * 50mm) or coordinator.Before mass spectrophotometry, can the eluent from extraction column be applied to analytic type LC post then, like HPLC post with on-line mode.Because the step that comprises in these chromatographic programs can connect with the robotization mode, so can the needs of in the analyte purge process operator being participated in be reduced to minimum.These characteristics can cause saving time and cost, and eliminate the possibility of operator error.

Detection through mass spectroscopy with quantitatively

In various embodiments, the vitamin D of deriving can be through the known any method of those skilled in the art and by ionization.Utilize mass spectrometer to carry out mass spectroscopy, mass spectrometer comprises that the sample that is used for the ionization classification is used for the further ion gun of the charged molecule of analysis with producing.For example the ionization of sample can be passed through electron ionization; Chemi-ionization; Electrospray ionization (ESI); Photoionization; APCI (APCI); Photoionization; Atmospheric pressure photoionization (APPI); Laser diode thermal desorption (LDTD); Fast atom bombardment (FAB); Liquid double ionization (LSI); Matrix assisted laser desorption ionization (MALDI); Field-ionization; Field desorption; Thermojet/plasma jet ionization; Surface-enhanced laser desorption ionization (SELDI); Inductively coupled plasma (ICP) and particle beam ionization are carried out.Those skilled in the art will understand, can be based on the selection with measured analyte, sample type, detector type, positive definite ionization methods such as selection to negative mode.

The vitamin D of deriving can be with the ionization of positive or negative mode.The vitamin D of deriving in a preferred embodiment, with the kation mode through APCI or LDTD and by ionization.

In the mass spectroscopy technology, after sample is by ionization, can analyze consequent positively charged or electronegative ion usually to measure mass-to-charge ratio.The suitable analyzer of measuring mass-to-charge ratio comprises four utmost point analyzers, ion trap analyzer and Time-of flight analyzer.Exemplary ion trap method is at Bartolucci, et al., and Rapid Commun.Mass Spectrom.2000 is described among the 14:967-73.

Several kinds of detection modes capable of using detect ion.For example, the ion of selection can be to be detected, promptly utilizes selected ion monitoring mode (SIM), or alternatively, can be monitored by quality transition or neutral forfeiture that collision induced dissociation produces, for example, multiple reaction monitoring (MRM) or selective reaction monitoring (SRM).Preferably, utilize four utmost point analyzers to measure mass-to-charge ratio.For example, in " four utmost points " or " quadrupole ion trap " instrument, ion experience in the vibration radio-frequency field and the DC electromotive force that between electrode, applies, the amplitude and the proportional power of mass-to-charge ratio of RF signal.Can select voltage and amplitude so that only have the length that the ion of specific mass-to-charge ratio experiences four utmost points, and all other ion deflection.Therefore, four utmost point instruments can serve as " mass filter " and " mass detector " of the ion that is injected into instrument.

People can improve the resolution of MS technology through utilizing " tandem mass spectrometry " or " MS/MS ".In this technology; Can the MS instrument, filter from the precursor ion (being also referred to as parent ion) that molecule (s) of interest produces; Broken subsequently precursor ion is analyzed it to produce one or more fragmentions (being also referred to as daughter ion or product ion) then in the 2nd MS program.Through carefully selecting precursor ion, the ion that only some analyte is produced is delivered to fragmentation cell, and the atomic collision with inert gas produces fragmention there.Because precursor ion and fragmention all produce with reproducible mode under given ionization/cracked condition, the MS/MS technology can provide very powerful analysis tool.For example, filtration/cracked combination can be used for eliminating interfering material, and can be at complex sample, as particularly useful in the biological sample.

The substitute mode of operating series mass spectrometer comprises product ion scanning and precursor ion scanning.Description for these modes of operation; Referring to, for example, E.Michael Thurman; Et al.; Chromatographic-Mass Spectrometric Food Analysis for Trace Determination of Pesticide Residues, the 8th chapter (AmadeoR.Fernandez-Alba compiles, and Elsevier 2005) (387).

Can be the result of analyte determination is related with the amount of analyte in the original sample through numerous method known in the art.For example, suppose careful control sampling and analytical parameters, can the table of the relative abundance of given ion with the absolute magnitude that this relative abundance is changed into initial molecule be compared.Alternatively, external standard can carry out with sample, makes typical curve based on the ion that produces from those standards.Utilize such typical curve, can the relative abundance of given ion be changed into the absolute magnitude of initial molecule.In some preferred implementation, interior mark is used to produce the typical curve of the amount of calculating vitamin D.Produce and utilize the method for such typical curve to be well known in the art, those of ordinary skill can be selected suitable interior mark.For example, in a preferred embodiment, one or more isotope-labeled vitamin D (for example, vitamin Ds ₂-[6,19,19]- ²H ₃And vitamin D ₃-[6,19,19]- ²H ₃) can be used as interior mark.Other numerous method that the amount of ion is associated with the amount of initial molecule will be known to those skilled in the art.

One or more steps automatic machinery capable of using of method carries out.In some embodiments, one or more purification steps are online to carry out, and more preferably all purifying and mass spectroscopy step can be carried out with on-line mode.

In some embodiments, like MS/MS, wherein with precursor ion separate be used for further cracked, collisional activation dissociate (CAD) usually be used to produce fragmention and be used for further detection.In CAD, precursor ion obtains energy through colliding with inert gas, and is broken through the process that is called " unimolecule decomposition " subsequently.Enough energy must be deposited in the precursor ion so that because the energy of vibration that increases, and some key in the ion can be ruptured.

In some preferred implementations, can utilize the vitamin D in MS/MS detection and/or the quantitative sample as follows.Sample can at first extract purifying through protein precipitation or hybrid protein matter deposition/liquid-liquid.Then, randomly with the vitamin D in the sample of purifying with Cookson type reagent, derive like PTAD.Sample with purifying carries out liquid chromatography then, on extraction column (like the TFLC post), is analytical column (like the HPLC post) after this extraction column preferably; Flow into the atomizer interface of MS/MS analyzer from the liquid flux of chromatographic column; Solvent/analyte mixture is converted to steam in the charged pipeline of the heating of interface.Through big voltage is applied to solvent/analyte mixture with the analyte (one or more) that contains in the solvent (vitamin D of for example, deriving or do not derive) ionization.When analyte withdraws from the charged pipeline of interface, solvent/analyte mixture atomizing, solvent evaporation stays analyte ions.Analyte ions, precursor ion for example, the aperture through instrument also gets into the one or four utmost point.Four utmost points 1 and 3 (Q1 and Q3) are mass filters, allow to select ion (that is, select respectively among Q1 and the Q3 " pioneer " ion and " fragment " ion) based on their mass-to-charge ratio (m/z).Four utmost points 2 (Q2) are the collision cells, and ion is broken there.The ion that mass spectrometric the one or four utmost point (Q1) selection has interested mass-to-charge ratio.Allow to have correct matter/precursor ion of lotus ratio gets into collision cell (Q2), and have any other mass-to-charge ratio unwanted ion and four utmost points side impact and be eliminated.Get into precursor ion and the neutral argon molecules bump and broken of Q2.The fragmention that produces gets into four utmost points 3 (Q3), and the vitamin D fragmention of deriving there or do not derive is selected, and other ion is excluded.

Method can comprise with kation mode or negative ion mode, the MS/MS that the preferred cationic mode is carried out.Utilize standard method well known in the art, those skilled in the art can identify one or more fragmentions that can be used for the particular precursor ion of the vitamin D of deriving of selection in four utmost points 3 (Q3).

When ion and detecting device collision, they produce electronic impulse, and it is converted into digital signal.Give computing machine with the data transfer that obtains, the ion counting that computer drawing is collected is to the figure of time.The mass spectrogram that produces is similar with the chromatogram that traditional HPLC-MS method produces.Can measure corresponding to the amplitude at area under the peak of specific ion or such peak and make it to be associated with the amount of interested analyte.In some embodiments, measure fragmention (one or more) and/or the TG-AUC of precursor ion or the amplitude at peak, to measure the amount of vitamin D.As stated, based on the peak of one or more ions of interior molecules standard, calibration standard curve capable of using converts the relative abundance of given ion to the absolute magnitude of original analysis thing.

Embodiment

Embodiment 1: hybrid protein matter deposition/liquid-liquid extracts and the Cookson type is derived

Patient serum sample is carried out the hybrid protein matter deposition/liquid-liquid extractive technique of following robotization.Gel barrier serum (Gel Barrier Serum) (that is the serum of, in serum separator tube, gathering) and edta plasma and heparin blood plasma have been established as that this mensuration accepts.

Perkin-Elmer Janus robot and TomTec Quadra Tower robot are used to make following programming automation.For each sample, 50 μ L serum are added in the hole of 96 orifice plates.In each hole, mark potpourri in the adding 25 μ L then and (comprise isotope-labeled vitamin D ₃-[6,19,19]- ²H ₃), and with the plate vortex.Add 75 μ L methyl alcohol then, carry out other vortex afterwards.Add 300 μ L ethyl acetate and 75 μ L water then, carry out other vortex, centrifugal afterwards, and the supernatant that obtains is transferred to 96 new orifice plates.

The liquid of the transfer in the 2 96 orifice plate is fully dry under the flowing nitrogen manifold.0.1mg/mL solution through the Cookson type derivating agent PTAD in the acetonitrile that adds from 100 μ L to each hole is realized deriving.Allow derivatization reaction to carry out about one hour, and make it cancellation through in reaction mixture, adding 100 μ L water.

Embodiment 2: extract vitamin D with liquid chromatography

Utilize the software of Aria OS V 1.5.1 or renewal to carry out the sample injection with Cohesive Technologies Aria TX-4 TFLC system.

TFLC system automation ground is injected into the aliquot of the sample of above-mentioned preparation and is filled with the online extraction column of oarse-grained Cohesive Technologies C8XL (50 μ m granularities, 005 * 50mm is from Cohesive Technologies, Inc.).Load sample is with the turbulization in extraction column inside under high flow rate.The vitamin D that this turbulent flow is guaranteed to derive in the post and bulky grain are optimized combine and excessive derivative reagent and fragment to the passage of refuse.

After the loading, water/ethanol elution gradient is eluted to analytical column with sample---Thermo Hypersil Gold Aq analytical column (5 μ m granularities, 50 * 2.1mm).The HPLC gradient is applied to analytical column so that other analyte that contains in vitamin D and the sample is separated.Mobile phase A is a water, and Mobile phase B is an ethanol.The HPLC gradient is since 35% organic gradient, and it rose to 99% in about 65 seconds.

Embodiment 3: detect and the vitamin D of quantitatively deriving through MS/MS

Utilize Finnigan TSQ Quantum Ultra MS/MS system (Thermo Electron Corporation) that the sample of top generation is carried out MS/MS.Following software program---all are all from Thermo Electron---uses in embodiment described herein: Quantum Tune Master V 1.5 or upgrade, Xcalibur V 2.07 or upgrade, LCQuan V 2.56 (Thermo Finnigan) or upgrade with ARIA OS v1.5.1 (Cohesive Technologies) or upgrade.Withdraw from the atomizer interface of the liquid flux/analyte stream of analytical column to the MS/MS analyzer.Solvent/analyte mixture is converted to steam in the pipeline of interface.Analyte in the solvent of atomizing is ionized by APCI.

Ion is through the one or four utmost point (Q1), and it selects to have respectively the vitamin D of mass-to-charge ratio 572.3 ± 0.5m/z and 560.3 ± 0.5m/z ₂And vitamin D ₃Precursor ion.Get into the ion and the argon gas collision of four utmost points 2 (Q2), produce fragment ion, it is sent to four utmost points 3 (Q3) and is used for further selection.The mass spectrometer setting is presented at table 1.Simultaneously, use interior mark, vitamin D ₃– [6,19,19]- ²H ₃Utilize the identical process of isotope dilution mass spectrometry.Under the collision energy of positive polarity and indication, be used to detect with quantitative quality transition in the process of checking and be presented at table 2.

Table 1. is used for the PTAD-vitamin D ₂, the PTAD-vitamin D ₃And vitamin D ₃– [6,19,19]- ²H ₃The mass spectrometer setting that (interior mark) (positive polarity) detects

The vitamin D that table 2.PTAD derives ₂, vitamin D ₃And vitamin D ₃– [6,19,19]- ²H ₃The quality transition of (interior mark) (positive polarity)

The PTAD-vitamin D ₂, the PTAD-vitamin D ₃, the PTAD-vitamin D ₃-[6,19,19]- ²H ₃The exemplary chromatogram of (interior mark) is presented at respectively among Figure 1A, 1B and the 1C.

Vitamin D in the serum sample ₂And vitamin D ₃The exemplary calibration curve of mensuration be presented at respectively among Fig. 2 A and the 2B.

Embodiment 4: The vitamin D MS/MS that derives detects the linearity of response

According to the method for embodiment 1-3, through vitamin D with high endogenous concentration ₂Or vitamin D ₃Dilute the dilution (in duplicate) of four serum set (pool) and analysis 75%, 50% and 25% and measure linearity.Sample can dilute with 1:4, has 102% average recovery rate, but allows the clinical reporting range (CRR) of 2-240ng/mL in the degree of accuracy limit value of 85%-115%CV.The measured value of these researchs and recovery percentages show are in table 3.

Table 3. shows the data of the linearity of response in the dilution range

Embodiment 5: Sensitivity for analysis: lower limit of quantitation (LLOQ) and detection limit value (LOD)

Lower limit of quantitation (LLOQ) is to measure the quantitatively significant point that becomes.In the response of the analyte of this LLOQ is discernible, discrete with reproducible, has degree of accuracy (that is the coefficient of variation (CV)) and 80% to 120% accuracy greater than 20%.According to the method for embodiment 1-3, have quadruplicate five times of the sample of known analyte concentration (2ng/mL, 5ng/mL, 10ng/mL, 15ng/mL, 20ng/mL, 40ng/mL and 60ng/mL) through mensuration, estimate repeatability then and measure LLOQ.Analysis to the data of collecting shows that for two kinds of analytes, the sample that has less than 2ng/mL concentration all produces the CVs less than 20%.Therefore, the LLOQ of every kind of analyte is confirmed as less than 2ng/mL.The PTAD-vitamin D ₂With the PTAD-vitamin D ₃The data that measure to produce of LLOQ be presented at respectively in the table 4 and 5.The CV of two kinds of analytes is presented among Fig. 3 the diagrammatic representation of concentration.

Table 4. vitamin D ₂The mensuration of lower limit of quantitation

Table 5. vitamin D ₃The mensuration of lower limit of quantitation

Detecting limit value (LOD) is such point, and at this point, measured value is greater than relative uncertainty and the 4 times of standard deviations (SD) when at random being defined as zero-dose.Selectivity is in sample, to exist analytical approach under the situation of other component to distinguish and the ability of quantifying analytes.According to the method for embodiment 1-3, analysis margin in 20 replicate determinations (replicate), and on statistics, analyze the area ratio that produces, record vitamin D ₂And vitamin D ₃LOD all be 0.4ng/mL.The data presentation of the collected LOD that is used for measuring every kind of analyte is at table 6.

Table 6. vitamin D ₂And vitamin D ₃The mensuration of detection limit value

The replicate determination numbering	NVD 2(response ratio)	NVD 3(response ratio)
			1	0.012	0.014
2	0.004	0.005
			3	0.008	0.011
4	0.005	0.004
			5	0.018	0.03
6	0.004	0.001
			7	0.007	0.006
8	0.006	0.002
			9	0.014	0.006
10	0.015	0.006
			11	0.003	0.008
12	0.005	0.004
			13	0.009	0.002
14	0.017	0.016
			15	0.007	0.005

16	0.006	0.012
			17	0.003	0.001
18	0.002	0.001
			19	0.011	0.001
20	0.023	0.009
			Mean value	0.009	0.007
SD	0.0058	0.0070
			CV	65.1	97.0
Mean value+4SD	0.032	0.035
			LOD	0.4ng/mL	0.4ng/mL

Embodiment 7: The specificity that detects

Method according to embodiment 1-3 is used vitamin D ₂, vitamin D ₃With the potential interference species (comprising vitamin D metabolism thing and related compound) of the incorporation several samples of preparation and analyze.The compound of having tested potential interference is listed in the table 7.According to the method for embodiment 1-3, the compound of test does not all have to show and vitamin D ₂Or vitamin D ₃The cross reactivity that detects.

But table 7. test interfere vitamin D ₂Or vitamin D ₃The compound that detects

The compound of test
	1,25(OH) 2 D 3
1,25(OH) 2 D 2
	1,25(OH) 2D 3-[6,19,19']- 2 H
1,25(OH) 2D 3-[26,26,26,27,27,27]- 2 H
	1,25(OH) 2D 2-[26,26,26,27,27,27]- 2H
25OHD 3
	25OHD 2
25OHD 3-IS-[6,19,19']- 2H
	25OHD 2-IS-[6,19,19']- 2H
25OHD 3-IS-[26,26,26,27,27,27]- 2H
	25OHD 2-IS-[26,26,26,27,27,27]- 2H
Vitamin D 3-[6,19,19']- 2H
	Vitamin D 2-[6,19,19']- 2H
Vitamin D 3-[26,26,26,27,27,27]- 2H
	Vitamin D 2-[26,26,26,27,27,27]- 2H
1-OH-D 3(Alfacalcidol)
	1-OH-D 2(Hectoral)

24,25(OH) 2 D 3
	25,26(OH) 2D 3
3-table-25OHD 3
	3-shows-1,25 (OH) 2D 3
Dihydrotachysterol
	1,25(OH) 2D 3-26, the 23-lactone
Paracalcitol (Paracalcitol) (Zemplar)
	Calcipotriene (Calcipotriene) (Dovonex)
The 7-dehydrocholesterol

Embodiment 8: Vitamin D ₂ And vitamin D ₃ Quantitative repeatability

(in the mensuration, intra-assay) variation is defined as the repeatability of measuring interior sample, and measures through measuring from 20 parallel sample of the sample of each in three QC set according to the method for embodiment 1-3 in batch.Respectively to vitamin D ₂And vitamin D ₃, the data presentation of from these are analyzed, collecting is in table 8 and 9.Analyte concentration in the QC set is for vitamin D ₂Be measured as 6.6ng/mL, 20.6ng/mL and 52.6ng/mL, for vitamin D ₃Be measured as 4.9ng/mL, 20.5ng/mL and 48.6ng/mL.The statistics that the result is carried out produces for vitamin D ₂The repeatability of three QC set 5.1%, 4.6% and 3.9% is for vitamin D ₃The repeatability of three QC set 6.4%, 4.0% and 4.5%.

Table 8. vitamin D ₂Variation within batch measure

Table 9. vitamin D ₃Variation within batch measure

(between mensuration, inter-assay) variation is defined as the repeatability (CV) of sample between the mensuration between will criticizing.According to the method for embodiment 1-3, but utilize the QC of the reporting range of three overlay measurements to gather---in 5 mensuration, estimate, to vitamin D ₂And vitamin D ₃Measure the batch variation (CV) of set.For vitamin D ₂, CV is measured as 6.7%, 5.6% and 4.0% respectively, has the mean concentration of 6.5ng/mL, 21.1ng/mL and 50.5ng/mL.For vitamin D ₃, CV is measured as 6.5%, 5.9% and 4.2% respectively, has the mean concentration of 4.7ng/mL, 20.8ng/mL and 46.8ng/mL.To vitamin D ₂And vitamin D ₃, analyze the data of collecting from these and be presented at respectively the table 10 and 11.All set satisfy≤the acceptable repeatability requirement of 15%CV.

Table 10. vitamin D ₂Batch variation measure

Table 11. vitamin D ₃Batch variation measure

Embodiment 9: Vitamin D ₂ The quantitative methods correlative study

Compare 20 samples that separate, to vitamin D through---being HPLC that band UV detects afterwards---according to embodiment 1-3 method according to tandem mass spectrum methods analyst described herein with off line extraction widely ₂Quantitatively carry out the method correlative study.For each method, sample is analyzed separately.Data return through linear regression and Deming and analyze.Correlation analysis is summarised in the table 12.

The correlation analysis relatively of table 12. method

Embodiment 10: The Study of Interference

Haemolysis disturbs:Through haemoglobin being mixed the vitamin D that comprises raising ₂And vitamin D ₃Serum gather the influence of estimating in the mensuration that haemolysis describes in embodiment 1-3.Fresh blood samples is centrifugal with the generation red blood cell (packed red blood cells) that concentrates.With cell reconstruct and freezing in deionized water to obtain cytolysis.Then this rough hemoglobin solutions is incorporated into set to produce the haemolysis sample of slight (100mg/dL) and moderate (500mg/dL).Method according among the embodiment 1-3 is analyzed sample in duplicate, and the result is compared and calculated difference number percent with the contrast aggregated result.Data presentation for any analyte, is compared with contrast, and haemoglobin mixes all not to be had>15% difference).Therefore, the sample of mild to moderate haemolysis is acceptable.For raw data, referring to table 13 (% difference=(it is uncorporated to mix De –)/uncorporated * 100%).

Table 13. haemolysis The Study of Interference

Jaundice is disturbed:Through cholerythrin being mixed the vitamin D that comprises raising ₂And vitamin D ₃Serum gather the influence of estimating in the mensuration that jaundice describes in embodiment 1-3.The bilirubin solution that concentrates is incorporated into set to produce the jaundice sample of slight (10mg/dL) and moderate (50mg/dL).Method according among the embodiment 1-3 is analyzed sample in duplicate, and with result and comparison of no jaundice aggregated result and accuracy in computation.Data presentation, two analytes all do not receive jaundice to influence (all values are all in the acceptable accuracy range of 85-115%).Therefore, the jaundice sample is acceptable.For raw data, referring to table 14 (% difference=(mix-uncorporated)/uncorporated * 100%).

Table 14. jaundice The Study of Interference

Pionemia is disturbed:Through Medulla sus domestica extract being mixed the vitamin D that comprises raising ₂And vitamin D ₃Serum gather the influence of estimating in the mensuration that pionemia describes in embodiment 1-3.Powdered lipid sample (Avanti Polar Lipids) is dissolved in each set to produce the piarhemia sample of slight (400mg/dL) and moderate (2000mg/dL).Method according among the embodiment 1-3 is analyzed sample in duplicate, and with result and contrast aggregated result comparison and accuracy in computation.Data presentation, two analytes all do not receive pionemia to influence (all values are all in the acceptable accuracy range of 85-115%).For raw data, referring to table 15 (% difference=(mix-uncorporated)/uncorporated * 100%).

Table 15. pionemia is disturbed (Medulla sus domestica extract) research

Also through Intralipid (Intralipid) emulsion is mixed the vitamin D that comprises evaluation ₂And vitamin D ₃Serum gather the influence of estimating in the mensuration that pionemia describes in embodiment 1-3.In the serum set, add Intralipid (20% emulsion) to produce the piarhemia sample of slight (400mg/dL) and moderate (2000mg/dL).Method according among the embodiment 1-3 is analyzed sample in duplicate, and with result and contrast aggregated result comparison and accuracy in computation.Data presentation, two analytes all do not receive pionemia to influence (all values are all in the acceptable accuracy range of 85-115%).For raw data, referring to table 16.

Table 16. pionemia is disturbed (Intralipid) research

Based on two pionemia experiments that utilize Medulla sus domestica extract and Intralipid, the piarhemia sample is acceptable.

Embodiment 11: Sample type research

Sample is collected four different containers from 10 sources.The Vacutainers that uses is Red-Top (serum tube of silicon-coated), SST (serum separator tube, it causes gel barrier serum), EDTA pipe and liquaemin pipe.

Trophism vitamin D according to these 40 samples of the methods analyst among the embodiment 1-3 ₃Result relatively is presented among Fig. 4.Data presentation, all four sample types all are fit to analyze.

Embodiment 12: Vitamin D ₃ The proof of normal ranges

According to the methods analyst of describing among the embodiment 1-3 from 140 patients' serum sample with quantitative vitamin D ₃The result is that scope does<2ngmL is to the vitamin D of about 63ng/mL ₃, 95% result drops into<2ngmL is to the scope of about 20ng/mL.The result of these analyses is presented in the table 17.

Table 17. vitamin D ₃Normal ranges research (with descending order)

Embodiment 13: Reclaim research

Through having the 25-hydroxy-vitamin D of elevated levels natively ₂Or 25-hydroxy-vitamin D ₃The analysis of sample mix and reclaim research, therefore also have some endogenous circulation vitamin Ds ₂Or vitamin D ₃Select six pairs of samples to be used for research.Also analyze in quadruplicate from five samples of every pair of sample (being known as sample A and sample B generically) preparation according to the method among the embodiment 1-3.Sample is corresponding to 100%A, 80%A-20%B, 50%A-50%B, 20%A-80%B and 100%B.The result who reclaims research is presented in the table 18 and 19.

Table 18. mixing sample reclaims research

* the value of measuring is the mean value that four aliquots are analyzed.

Table 19. mixing sample reclaims the result of study general introduction

Embodiment 14: Vitamin D ₂ And vitamin D ₃ The exemplary spectrum analyzed of MS/MS

From vitamin D ₂, vitamin D ₂– [6,19,19]- ²H ₃And vitamin D ₂– [26,26,26,27,27,27]- ²H ₆The exemplary Q1 scanning spectrum of tandem mass spectrum analysis be presented at respectively among Fig. 5 A, 6A and the 7A.Be injected into Finnigan TSQ Quantum Ultra MS/MS system (Thermo Electron Corporation) through the standard solution that directly will contain interested analyte and carry out these analyses.Through with 80% acetonitrile of 800 μ L/min, have 0.1% formic acid 20% water through the HPLC post, the liquid phase chromatogram flow phase is simulated at the upper reaches that analyte imports.As above described analyte passes through APCI by ionization.Whole m/z scope scanning Q1 through about 300 to 450 collects spectrum.

From vitamin D ₂, vitamin D ₂– [6,19,19]- ²H ₃And vitamin D ₂– [26,26,26,27,27,27]- ²H ₆In the exemplary products ion scan that produces of the different precursor ions of each two be presented on respectively among Fig. 5 B-C, 6B-C and the 7B-C.The precursor ion of selecting among the Q1 is presented at table 20 with the collision energy that is used for producing these product ions spectrums.

For vitamin D ₂Quantitative exemplary MRM changes and comprises the precursor ion with m/z of about 397.2 is broken into the product ion with m/z of about 159.0; The precursor ion that will have about 379.2 m/z is broken into the product ion with m/z of about 158.9.For vitamin D ₂– [6,19,19]- ²H ₃Quantitative exemplary MRM changes and comprises the precursor ion with m/z of about 400.2 is broken into the product ion with m/z of about 147.0; The precursor ion that will have about 382.2 m/z is broken into the product ion with m/z of about 312.2.For vitamin D ₂– [26,26,26,27,27,27]- ²H ₆Quantitative exemplary MRM changes and comprises the precursor ion with m/z of about 403.2 is broken into the product ion with m/z of about 159.1; The precursor ion that will have about 385.2 m/z is broken into the product ion with m/z of about 159.0.Yet like finding in the product ion scanning among Fig. 5 B-C, 6B-C and the 7B-C, several kinds of other product ions produce after precursor ion is cracked.Select other product ion in those product ions that can from Fig. 5 B-C, 6B-C and 7B-C, show, with replacement or increase exemplary fragmention.For example, by vitamin D with m/z of about 397.2 ₂The other product ion of the cracked generation of precursor ion comprises the ion with about m/z of 146.9,133.1 and 121.0.By vitamin D with m/z of about 379.2 ₂The exemplary other product ion of the cracked generation of precursor ion comprises the ion with about m/z of 283.2,187.3 and 175.2.

Table 20. vitamin D ₂, vitamin D ₂– [6,19,19]- ²H ₃And vitamin D ₂– [26,26,26,27,27,27]- ²H ₆Cracked precursor ion and collision cell energy

From vitamin D ₃, vitamin D ₃– [6,19,19]- ²H ₃And vitamin D ₃– [26,26,26,27,27,27]- ²H ₆The exemplary Q1 scanning spectrum of tandem mass spectrum analysis be presented at respectively among Fig. 8 A, 9A and the 10A.Be injected into Finnigan TSQ Quantum Ultra MS/MS system (Thermo Electron Corporation) through the standard solution that directly will contain interested analyte and carry out these analyses.Through with 80% acetonitrile of 800 μ L/min, have 0.1% formic acid 20% water through the HPLC post, the liquid phase chromatogram flow phase is simulated at the upper reaches that analyte imports.Through collecting spectrum at whole about 300 to 450 m/z scope scanning Q1.

From vitamin D ₃, vitamin D ₃– [6,19,19]- ²H ₃And vitamin D ₃– [26,26,26,27,27,27]- ²H ₆In the exemplary products ion scan that produces of the different precursor ions of each two be presented on respectively among Fig. 8 B-C, 9B-C and the 10B-C.The precursor ion of selecting among the Q1 is presented at table 21 with the collision energy that is used for producing these product ions spectrums.

For vitamin D ₃Quantitative exemplary MRM changes and comprises the precursor ion with m/z of about 385.2 is broken into the product ion with m/z of about 147.0; The precursor ion that will have about 367.2 m/z is broken into the product ion with m/z of about 159.0.For vitamin D ₃– [6,19,19]- ²H ₃Quantitative exemplary MRM changes and comprises the precursor ion with m/z of about 388.2 is broken into the product ion with m/z of about 147.0; The precursor ion that will have about 370.2 m/z is broken into the product ion with m/z of about 160.2.For vitamin D ₃– [26,26,26,27,27,27]- ²H ₆Quantitative exemplary MRM changes and comprises the precursor ion with m/z of about 391.2 is broken into the product ion with m/z of about 159.1; The precursor ion that will have about 373.2 m/z is broken into the product ion with m/z of about 159.0.Yet like finding in the product ion scanning among Fig. 8 B-C, 9B-C and the 10B-C, several kinds of other product ions produce after precursor ion is cracked.Select other product ion in those product ions that can from Fig. 8 B-C, 9B-C and 10B-C, show, with replacement or increase exemplary fragmention.For example, by vitamin D with m/z of about 385.2 ₃The other product ion of the cracked generation of precursor ion comprises the ion with about m/z of 159.0,133.1 and 107.1.By vitamin D with m/z of about 367.2 ₃The other exemplary products ion of the cracked generation of precursor ion comprises the ion with about m/z of 172.9,145.0 and 119.1.

Table 21. vitamin D ₃, vitamin D ₃– [6,19,19]- ²H ₃And vitamin D ₃– [26,26,26,27,27,27]- ²H ₆Cracked precursor ion and collision cell energy

Embodiment 15: The vitamin D that PTAD derives ₂ And vitamin D ₃ The exemplary light analyzed of MS/MS Spectrum

Aliquot through handle the stock solution of each analyte with the PTAD in the acetonitrile prepares vitamin D ₂, vitamin D ₂– [6,19,19]- ²H ₃, vitamin D ₂– [26,26,26,27,27,27]- ²H ₆, vitamin D ₃, vitamin D ₃– [6,19,19]- ²H ₃And vitamin D ₃– [26,26,26,27,27,27]- ²H ₆The PTAD derivant.Allow derivatization reaction to carry out about one hour, and make it cancellation through in reaction mixture, adding entry.The analyte of deriving according to the process analysis of describing among the top embodiment 2-3 then.

Come self-contained PTAD-vitamin D ₂, the PTAD-vitamin D ₂– [6,19,19]- ²H ₃With the PTAD-vitamin D ₂– [26,26,26,27,27,27]- ²H ₆The exemplary Q1 scanning spectrum of sample analysis be presented at respectively among Figure 11 A, 12A and the 13A.Be injected into Finnigan TSQ Quantum Ultra MS/MS system (Thermo Electron Corporation) through the standard solution that directly will contain interested analyte and carry out these analyses.Through with 80% acetonitrile of 800 μ L/min, have 0.1% formic acid 20% water through the HPLC post, the liquid phase chromatogram flow phase is simulated at the upper reaches that analyte imports.As above through the APCI ionization of analytes with describing.Spectrum is collected through the whole m/z scope scanning Q1 about 500 to 620.

From the PTAD-vitamin D ₂, the PTAD-vitamin D ₂– [6,19,19]- ²H ₃With the PTAD-vitamin D ₂– [26,26,26,27,27,27]- ²H ₆In the exemplary products ion scan that produces of each precursor ion be presented on respectively among Figure 11 B, 12B and the 13B.The precursor ion of selecting among the Q1 is presented at table 22 with the collision energy that is used for producing these product ions spectrums.

For the PTAD-vitamin D ₂Quantitative exemplary MRM changes and comprises the precursor ion with m/z of about 572.2 is broken into the product ion with m/z of about 297.9.For the PTAD-vitamin D ₂– [6,19,19]- ²H ₃Quantitative exemplary MRM changes and comprises the precursor ion with m/z of about 575.2 is broken into the product ion with m/z of about 301.0.For the PTAD-vitamin D ₂– [26,26,26,27,27,27]- ²H ₆Quantitative exemplary MRM changes and comprises the precursor ion with m/z of about 578.2 is broken into the product ion with m/z of about 297.9.Yet like finding in the product ion scanning among Figure 11 B, 12B and the 13B, several kinds of other product ions produce after precursor ion is cracked.Select other product ion in those product ions that can from Figure 11 B, 12B and 13B, show, with replacement or increase exemplary fragmention.For example, the PTAD-vitamin D that has about 572.2 m/z ₂The other product ion of the cracked generation of precursor ion comprises the ion with m/z of about 280.1.

Table 22.PTAD-vitamin D ₂, the PTAD-vitamin D ₂– [6,19,19]- ²H ₃With the PTAD-vitamin D ₂– [26,26,26,27,27,27]- ²H ₆Cracked precursor ion and collision cell energy

Analyte	Precursor ion (m/z)	Collision cell energy (V)
			The PTAD-vitamin D 2	572.2	15
The PTAD-vitamin D 2-[6,19,19]- 2H 3	575.2	15
			The PTAD-vitamin D 2-[26,26,26,27,	578.2	15

27,27]- 2H 6

Come self-contained PTAD-vitamin D ₃, the PTAD-vitamin D ₃– [6,19,19]- ²H ₃With the PTAD-vitamin D ₃– [26,26,26,27,27,27]- ²H ₆The exemplary Q1 scanning spectrum of sample analysis be presented at respectively among Figure 14 A, 15A and the 16A.Be injected into Finnigan TSQ Quantum Ultra MS/MS system (Thermo Electron Corporation) through the standard solution that directly will contain interested analyte and carry out these analyses.Through with 80% acetonitrile of 800 μ L/min, have 0.1% formic acid 20% water through the HPLC post, the liquid phase chromatogram flow phase is simulated at the upper reaches that analyte imports.Whole m/z scope scanning Q1 through about 500 to 620 collects spectrum.

From the PTAD-vitamin D ₃, the PTAD-vitamin D ₃– [6,19,19]- ²H ₃With the PTAD-vitamin D ₃– [26,26,26,27,27,27]- ²H ₆In the exemplary products ion scan that produces of each precursor ion be presented on respectively among Figure 14 B, 15B and the 16B.The precursor ion of selecting among the Q1 is presented at table 23 with the collision energy that is used for producing these product ions spectrums.

For the PTAD-vitamin D ₃Quantitative exemplary MRM changes and comprises the precursor ion with m/z of about 560.2 is broken into the product ion with m/z of about 298.0.For the PTAD-vitamin D ₃– [6,19,19]- ²H ₃Quantitative exemplary MRM changes and comprises the precursor ion with m/z of about 563.2 is broken into the product ion with m/z of about 301.0.For the PTAD-vitamin D ₃– [26,26,26,27,27,27]- ²H ₆Quantitative exemplary MRM changes and comprises the precursor ion with m/z of about 566.2 is broken into the product ion with m/z of about 298.0.Yet like finding in the product ion scanning among Figure 14 B, 15B and the 16B, several kinds of other product ions produce after precursor ion is cracked.Select other product ion in those product ions that can from Figure 14 B, 15B and 16B, show, with replacement or increase exemplary fragmention.For example, the PTAD-vitamin D that has about 560.2 m/z ₃The other product ion of the cracked generation of precursor ion comprises the ion with m/z of about 280.0.

Table 23.PTAD-vitamin D ₃, the PTAD-vitamin D ₃– [6,19,19]- ²H ₃With the PTAD-vitamin D ₃– [26,26,26,27,27,27]- ²H ₆Cracked precursor ion and collision cell energy

The content of the information that the article that this paper mentions or quotes, patent and patented claim and all other files and electronic form can get all is merged in it at this by reference, its degree with like each separately open indicated particularly and individually be merged in by reference identical.The applicant keeps will have any and all material of shape file and e-file and the right that information is physically incorporated this application into from any such article, patent, patented claim or other.

The method that this paper describes illustratively can suitably be implemented under the situation that lacks the not concrete disclosed any composition of this paper or a plurality of composition, restriction or a plurality of restrictions.Therefore, for example, term " comprises (comprising) ", " comprising (including) ", " containing " etc. should be enlarged, and ground is understood and not restriction.In addition, term used herein is described rather than the term of restriction with expressing to be used as, is not desirably in to get rid of in the use of such term and expression to show and the feature of description or any equivalent of its part.Will be appreciated that, be possible in the various scopes that are modified in requirement of the present invention protection.Therefore; Be to be understood that; Though the present invention is open by particularly with optional characteristic through preferred implementation, the disclosed modification of the present invention that wherein embodies of this paper and change and can occur to those skilled in the art, such modification and change are considered in this scope of invention.

The present invention here broadly and is prevailingly described.Fall into general disclosed narrower kind and each of subclass classification and also form the part of method.This comprises the general description of method, and condition precedent or negativity restriction are to remove any theme in the subordinate, no matter whether the material of deletion is here specifically described.

Other embodiment is in appended claim.In addition, when the characteristic of method or aspect are described with the form of Ma Kushi group, those skilled in the art will recognize that therefore the present invention also is described with the form of the Asia group of any single composition of Ma Kushi group or composition.

Claims (48)

1. through vitamin D in the tandem mass spectrometry working sample ₂The method of amount, said method comprises step:

(i) make vitamin D from sample ₂Experience ionization source being suitable for producing under one or more conditions through the detectable precursor ion of mass spectroscopy, said precursor ion is selected from the ion with mass-to-charge ratio (m/z) of 397.2 ± 0.5 or 379.2 ± 0.5;

(ii) broken at least a said precursor ion is to produce one or more through the detectable fragmention of mass spectroscopy;

(iii) through mass spectrometric determination step (i) with (ii) in the amount of one or more said ions of generation; With

(iv) make the (iii) middle vitamin D of measuring of step ₂Vitamin D in the existence of ion and the said sample ₂Existence be associated,

If wherein the precursor ion of said fragmentation comprises the ion with m/z of 397.2 ± 0.5; Said fragmention comprises that one or more are selected from the ion of the ion with m/z of 159.0 ± 0.5,146.9 ± 0.5,133.1 ± 0.5 and 121.0 ± 0.5; And if the precursor ion of said fragmentation comprises the ion with m/z of 379.2 ± 0.5, said fragmention comprises that one or more are selected from the ion of the ion with m/z of 283.2 ± 0.5,187.3 ± 0.5,175.2 ± 0.5 and 159.0 ± 0.5.

2. the described method of claim 1, the precursor ion of wherein said fragmentation are the ions with m/z of 397.2 ± 0.5.

3. the described method of claim 1, the precursor ion of wherein said fragmentation are the ions with m/z of 379.2 ± 0.5.

4. each described method among the claim 1-3 wherein makes said sample before ionization, experience extraction column.

5. the described method of claim 4, wherein said extraction column is SPE (SPE) post.

6. the described method of claim 4, wherein said extraction column is turbulent flow liquid chromatography (TFLC) post.

7. each described method among the claim 4-6 wherein makes said sample other undergoing analysis post before ionization.

8. the described method of claim 7, wherein said analytical column is high performance liquid chromatography (HPLC) post.

9. the described method of claim 7, wherein said extraction column is connected with on-line mode with the said ionization source of analytical column with step (i).

10. each described method among the claim 1-9, wherein said ionization source is APCI (APCI) source.

11. each described method among the claim 1-10, wherein said tandem mass spectrometry carries out as multiple reaction monitoring, precursor ion scanning or product ion scanning.

12. each described method among the claim 1-11 further comprises the vitamin D that detects in the said sample ₃

13. the described method of claim 12, wherein said vitamin D ₂And vitamin D ₃By ionization simultaneously.

14. each described method among the claim 1-14, wherein said sample comprises biological sample.

15. the described method of claim 14, wherein said biological sample be from the mankind, and be said when human when taking from, the said vitamin D of measuring in the said sample ₂Amount be the amount that exists in the said sample.

16. each described method among the claim 14-15, wherein said sample comprises serum or blood plasma.

17. through vitamin D in the tandem mass spectrometry working sample ₃The method of amount, said method comprises step:

(i) make vitamin D from sample ₃Experience ionization source being suitable for producing under one or more conditions through the detectable precursor ion of mass spectroscopy, said precursor ion is selected from the ion with mass-to-charge ratio (m/z) of 385.2 ± 0.5 or 367.2 ± 0.5;

(ii) broken at least a said precursor ion is to produce one or more through the detectable fragmention of mass spectroscopy;

(iii) through mass spectrometric determination step (i) with (ii) in the amount of one or more said ions of generation; With

(iv) make the (iii) middle vitamin D of measuring of step ₂Vitamin D in the existence of ion and the said sample ₃Existence be associated,

If wherein the precursor ion of said fragmentation comprises the ion with m/z of 385.2 ± 0.5; Said fragmention comprises that one or more are selected from the ion of the ion with m/z of 159.0 ± 0.5,147.0 ± 0.5,133.1 ± 0.5 and 107.1 ± 0.5; And if the precursor ion of said fragmentation comprises the ion with m/z of 367.2 ± 0.5, said fragmention comprises that one or more are selected from the ion of the ion with m/z of 172.2 ± 0.5,145.0 ± 0.5 and 119.1 ± 0.5.

18. the described method of claim 17, the precursor ion of wherein said fragmentation are the ions with m/z of 385.2 ± 0.5.

19. the described method of claim 17, the precursor ion of wherein said fragmentation are the ions with m/z of 367.2 ± 0.5.

20. each described method among the claim 17-19 wherein makes said sample before ionization, experience extraction column.

21. the described method of claim 20, wherein said extraction column are SPE (SPE) posts.

22. the described method of claim 20, wherein said extraction column are turbulent flow liquid chromatography (TFLC) posts.

23. each described method among the claim 20-22 wherein makes said sample other undergoing analysis post before ionization.

24. the described method of claim 23, wherein said analytical column are high performance liquid chromatography (HPLC) posts.

25. each described method among the claim 23-24, wherein said extraction column is connected with on-line mode with the said ionization source of analytical column with step (i).

26. each described method among the claim 17-25, wherein said ionization source are APCI (APCI) sources.

27. each described method among the claim 17-26, wherein said tandem mass spectrometry carries out as multiple reaction monitoring, precursor ion scanning or product ion scanning.

28. each described method among the claim 17-27 further comprises the vitamin D that detects in the said sample ₂

29. the described method of claim 28, wherein said vitamin D ₂And vitamin D ₃By ionization simultaneously.

30. each described method among the claim 17-29, wherein said sample comprises biological sample.

31. the described method of claim 30, wherein said biological sample be from the mankind, and be said when human when taking from, the said vitamin D of measuring in the said sample ₃Amount be the amount that exists in the said sample.

32. each described method among the claim 30-31, wherein said sample comprises serum or blood plasma.

33. through vitamin D in the tandem mass spectrometry working sample ₂And vitamin D ₃The method of amount, said method comprises step:

(i) make vitamin D in the sample ₂And vitamin D ₃Be suitable for producing one or more through the detectable vitamin D of mass spectroscopy ₂Precursor ion passes through the detectable vitamin D of mass spectroscopy with one or more ₃Experience ionization source under the condition of precursor ion, said vitamin D ₂Precursor ion is selected from the ion with mass-to-charge ratio (m/z) of 397.2 ± 0.5 and 379.2 ± 0.5, said vitamin D ₃Precursor ion is selected from the ion with mass-to-charge ratio (m/z) of 385.2 ± 0.5 and 367.2 ± 0.5;

(ii) broken at least a said vitamin D ₂Precursor ion is to produce one or more through the detectable vitamin D of mass spectroscopy ₂Fragmention;

If the vitamin D of said fragmentation wherein ₂Precursor ion comprises the ion with m/z of 397.2 ± 0.5, so said vitamin D ₂Fragmention comprises that one or more are selected from the ion of the ion with m/z of 159.0 ± 0.5,146.9 ± 0.5,133.1 ± 0.5 and 121.0 ± 0.5, and if the vitamin D of said fragmentation ₂Precursor ion comprises the ion with m/z of 379.2 ± 0.5, so said vitamin D ₂Fragmention comprises that one or more are selected from and have 283.2 ± 0.5, the ion of the ion of 187.3 ± 0.5,175.2 ± 0.5 and 159.0 ± 0.5 m/z;

(iii) broken at least a said vitamin D ₃Precursor ion is to produce one or more through the detectable vitamin D of mass spectroscopy ₃Fragmention; With

If the vitamin D of said fragmentation wherein ₃Precursor ion comprises the ion with m/z of 385.2 ± 0.5, so said vitamin D ₃Fragmention comprises that one or more are selected from the ion of the ion with m/z of 159.0 ± 0.5,147.0 ± 0.5,133.1 ± 0.5 and 107.1 ± 0.5, and if the vitamin D of said fragmentation ₃Precursor ion comprises the ion with m/z of 367.2 ± 0.5, so said vitamin D ₃Fragmention comprises that one or more are selected from the ion of the ion with m/z of 172.2 ± 0.5,145.0 ± 0.5 and 119.1 ± 0.5;

(iv) through mass spectrometric determination step (i), one or more said vitamin Ds of producing in (ii) and (iii) ₂And vitamin D ₃The amount of ion; With

(v) make the (iv) middle said vitamin D of measuring of step ₂And vitamin D ₃Vitamin D in the amount of ion and the said sample ₂And vitamin D ₃Amount be associated.

34. the described method of claim 33, the vitamin D of wherein said fragmentation ₂Precursor ion is the ion with m/z of 397.2 ± 0.5.

35. the described method of claim 33, the vitamin D of wherein said fragmentation ₂Precursor ion is the ion with m/z of 379.2 ± 0.5.

36. each described method among the claim 33-35, the vitamin D of wherein said fragmentation ₃Precursor ion is the ion with m/z of 385.2 ± 0.5.

37. each described method among the claim 33-35, the vitamin D of wherein said fragmentation ₃Precursor ion is the ion with m/z of 367.2 ± 0.5.

38. each described method among the claim 33-37 wherein makes said sample before ionization, experience extraction column.

39. the described method of claim 38, wherein said extraction column are SPE (SPE) posts.

40. the described method of claim 38, wherein said extraction column are turbulent flow liquid chromatography (TFLC) posts.

41. each described method among the claim 40-41 wherein makes said sample other undergoing analysis post before ionization.

42. the described method of claim 41, wherein said analytical column are high performance liquid chromatography (HPLC) posts.

43. each described method among the claim 40-41, wherein said extraction column is connected with on-line mode with the said ionization source of analytical column with step (i).

44. each described method among the claim 33-43, wherein said ionization source are APCI (APCI) sources.

45. each described method among the claim 33-44, wherein said tandem mass spectrometry carries out as multiple reaction monitoring, precursor ion scanning or product ion scanning.

46. each described method among the claim 33-45, wherein said sample comprises biological sample.

47. the described method of claim 46, wherein said biological sample be from the mankind, and be said when human when taking from, the said vitamin D of measuring in the said sample ₂And vitamin D ₃Amount be the amount that exists in the said sample.

48. each described method among the claim 46-47, wherein said sample comprises serum or blood plasma.

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